Polysialylated neural cell adhesion molecule is involved in the neuroplasticity induced by axonal injury in the avian ciliary ganglion

We demonstrated previously in the quail ciliary ganglion, that the immunoreactivity for the neural cell adhesion molecule labeling the postsynaptic specializations of intraganglionic synapses decreases when synaptic remodeling is induced by crushing the postganglionic ciliary nerves. Here we show, in the same experimental conditions, that the immunolabeling for its polysialylated non-stabilizing isoform, which promotes cell plasticity, increases at these subcellular compartments. In control ganglia, poor immunolabeling for the polysialylated neural cell adhesion molecule was occasionally observed surrounding the soma of the ciliary neurons, in correspondence with the calyciform presynaptic ending and the perineuronal satellite cells sheath. At the electron microscope, several neuronal compartments, including some postsynaptic specializations, somatic spines and multivesicular bodies, were immunopositive. Three to six days after ciliary nerve crush, both the number of ciliary neurons labeled for the polysialylated neural cell adhesion molecule and the intensity of their immunolabeling increased markedly. Electron microscopy revealed that, in parallel to the injury-induced detachment of the preganglionic boutons, numerous postsynaptic specializations were found to be immunopositive. Twenty days later, when intraganglionic connections were re-established, polysialylated neural cell adhesion molecule immunoreactivity was comparable to that observed in control ganglia. The increase in immunolabeling also involved the other neuronal compartments mentioned above, the perineuronal satellite cells and the intercellular space between these and the ciliary neurons.From these results we suggest that the switch, at the postsynaptic specializations, between the neural cell adhesion molecule and its polysialylated form may be among the molecular changes occurring in axotomized neurons leading to injury-induced synaptic remodeling. Moreover, from the increase in polysialylated neural cell adhesion molecule immunolabeling, observed at the somatic spines and at the interface between neurons and perineuronal satellite cells, we suggest that this molecule may be involved not only in synaptic remodeling, but also in other more general aspects of injury-induced neuronal plasticity.     

Immunohistochemical localization of the neural cell adhesion molecule in the rat sciatic nerve.

The neural cell adhesion molecule (NCAM) has been widely studied in the early embryonal development of the nervous system. The data about NCAM distribution in the peripheral nerves during postnatal life are scant and some controversial. In the present study, the NCAM localization in the sciatic nerves of 15-day-old Wistar rats has been studied. Semi-thin sections of the nerves were immunotested with a polyclonal antibody (Santa Cruz Biotechnology) that recognizes rat NCAM. The antibody was visualized with donkey anti-goat IgG, conjugated to 12 nm colloidal gold, and silver amplification. In the myelinated nerve fibres, the immunoreactivity was associated with the axons, mainly with their plasma membrane, which was unstained in the nodes of Ranvier. The myelin sheaths and the myelinating Schwann cells were negative. The extracellular matrix and the bundles of non-myelinated nerve fibres were immunopositive.     

Development of cholinergic traits in the quail ciliary ganglion: expression of choline acetyltransferase-like immunoreactivity

The avian ciliary ganglion is a parasympathetic ganglion derived from the neural crest whose neurons provide cholinergic innervation to the eye. Here, we describe the time course of appearance and the morphology of cholinergic cells in the ciliary ganglion, as assessed by antibodies against choline acetyltransferase. Choline acetyltransferase immunoreactivity was first observed in 5.5-day-old quail embryos, 1 day after condensation of the ciliary ganglion. Both the intensity of choline acetyltransferase immunoreactivity and size of the choline acetyltransferase-immunoreactive cells increased with ganglionic age. By 12 days, a second population of choline acetyltransferase-immunoreactive cells, possibly corresponding to choroid neurons, was observed whose cells were smaller and less intensely stained than earlier differentiating choline acetyltransferase-immunoreactive cells. The percentage of choline acetyltransferase-immunoreactive cells was initially high, constituting approximately 50% of the total cell population. As a function of time, the proportion of cholinergic cells decreased, probably due to proliferation of non-neuronal cells and naturally-occurring cell death. Our results confirm the existence of two morphologically distinct populations of cholinergic neurons in the avian ciliary ganglion and demonstrate that these neuronal subpopulations express choline acetyltransferase immunoreactivity at different times in development. Because choroid neurons innervate their targets later than ciliary neurons, this finding is consistent with the hypothesis that target interactions regulate expression of choline acetyltransferase.     

Nerve dependent regulation of neural cell adhesion molecule expression in skeletal muscle

The expression of neural cell adhesion molecule was analysed by indirect immunofluorescence on adult mouse skeletal muscle subjected to a variety of experimental lesions. Adult mouse muscle does not express neural cell adhesion molecule at the sarcolemma. However, following denervation there is a rapid rise in neural cell adhesion molecule levels; this is initially in the cytoplasm of the myofibres but by 18 days there is intense reactivity at the sarcolemma. A nerve crush lesion was used to show that the increase in neural cell adhesion molecule levels following denervation is accompanied by a switch-off of neural cell adhesion molecule expression following reinnervation. Paralysis of skeletal muscle by botulinum toxin injection is sufficient to activate neural cell adhesion molecule expression. As paralysis of skeletal muscle by botulinum toxin is not accompanied by activation of muscle satellite cells or degeneration products of nerve or myelin, it suggests that the observed levels of neural cell adhesion molecule are synthesised by myofibres. As the expression of neural cell adhesion molecule in these lesions parallels the ability of skeletal muscle to accept innervation it is possible that neural cell adhesion molecule acts as a molecular cue at the sarcolemma in regulating synaptogenesis.     

Epithelial cell adhesion molecule expression in pituitary adenomas: an immunohistochemical study

An important amount of data correlating the expression of epithelial cell adhesion molecule (Ep-CAM) with cellular proliferation and de-differentiation could directly contribute to carcinogenesis. The aim of this study is to evaluate prognosis relevance of Ep-CAM expression in a group of pituitary adenomas. Epithelial cell adhesion molecule, proliferating cell nuclear antigen, and microvascular density labeling indices in pituitary adenomas were determined by immunohistochemistry on tissue samples obtained from each adenoma after surgery. We evaluated 45 adenomas. Sixty-two percent were nonsecretor adenomas and 37.8% were secretor tumors. Immunohistochemistry was scored for immunoexpression of Ep-CAM (cytoplasmic, membrane, and mixed pattern). Proliferating cell nuclear antigen and vascular density (CD34) labeling indices were assessed. Statistical significance was observed between Ep-CAM cytoplasmic immunoreactions (P = .000) and higher proliferating cell nuclear antigen (P = .001) in secretor adenomas compared with nonsecretor tumors. Vascular density labeling indices did not show statistical significance. Therefore, Ep-CAM could be evaluated to distinguish secretor and nonsecretor pituitary adenomas. These suggest that the markers could predict the growth potential of individual pituitary adenomas.     

Effects of minocycline and tetracycline on retinal ganglion cell survival after axotomy

In the present study, we compared the in vivo neuroprotective efficacy of intraperitoneally administered tetracycline and minocycline to enhance the survival of retinal ganglion cells (RGCs) following unilateral axotomy of the adult rat optic nerve. We also examined the effects of the tetracycline drugs on the activation of retinal microglia. RGCs in retinal whole-mounts were visualized by retrograde labeling with fluorogold. The presence of activated microglia was confirmed immunohistochemically using OX-42 monoclonal antibodies. Optic nerve axotomy produced RGC death and increased activation of microglia. No significant RGC loss was seen prior to 5 days and approximately 50% and 80-90% cell loss occurred at 7 and 14 days, respectively. Examination of the effects of tetracycline and minocycline on RGC survival at 7 days post-axotomy, revealed increased numbers of RGCs in minocycline-treated animals (75% of non-axotomized control) compared with vehicle-only (52% of control) and tetracycline-treated (58% of control) animals. The densities of RGCs (RGCs/mm2+/-S.D.) for control, vehicle-, tetracycline- and minocycline-treated axotomized animals were 1996+/-81, 1029+/-186, 1158+/-190 and 1497+/-312, respectively. The neuroprotective effect of minocycline seen at 7 days was transient, since RGCs present in minocycline-treated animals at 14 days post-axotomy (281+/-43, 14% of control) were not significantly different to vehicle-treated animals (225+/-47, 11% of control). OX-42 staining of activated retinal microglia was reduced in tetracycline- and minocycline-treated axotomized animals compared with axotomized animals receiving vehicle-only. These results demonstrate that systemic administration of the second-generation tetracycline derivative, minocycline, delays the death of axotomized RGCs by a mechanism that may be associated with inhibition of microglia activation. The neuroprotective efficacy of minocycline following optic nerve axotomy was superior to that of tetracycline.     

Neuronal ultrastructure and somatostatin immunolocalization in the ciliary ganglion of chicken and quail

Summary Ciliary and choroid neurons of the avian ciliary ganglion innervate different targets in the eye bulb. By light microscopic immunocytochemistry, somatostatin (SOM) has been localized to a subset of ganglionic neurons believed to be, for the most part, choroid neurons. Although several studies have been published on the physiology, afferent and efferent innervation, and response to experimental injury of this population of cells, their morphological features are still unclear. This has led us to perform a fine structural and immunocytochemical study on the ciliary ganglia of adult chickens and quails to provide the first thorough characterization of the choroid neurons and to analyze whether or not they can be unequivocally identified by expression of SOM. Here, we show that standard and immuno-electron microscopy provide firm criteria for the distinction of ciliary and choroid neurons, whose populations overlap in cell size and territory of distribution. The satellite cell sheaths form compact myelin lamellae around ciliary neurons and flattened processes around choroid neurons. Moreover, ciliary neurons are innervated by a larger number of boutons than choroid neurons. Chicken ciliary neurons are invested by boutons only over one pole of the cell body, while their quail counterparts have an almost complete shell of presynaptic boutons over the entire cell body. Ciliary neurons form mixed synaptic junctions (chemical and electrical), while choroid neurons form only chemical synapses. Crest synapses are present in ciliary neurons of both species. Nematosomes occur in both ciliary and choroid neurons. Choroid neurons contain a larger complement of large dense core vesicles than ciliary neurons and their Golgi apparatuses are more prominent. In the light microscope, somatostatin-immunostaining appears noticeably different in the two species: mostly granular in the chicken and skein-shaped in the quail. Immuno-electron microscopy reveals that somatostatin-like immunoreactivity is localized to Golgi apparatus and large dense core vesicles. Somatostatin is expressed by all the choroid neurons, but not by the ciliary neurons. This neuropeptide is, therefore, a true cell population marker.     

Surface expression of neural cell adhesion molecule in cultured bovine paraneurones: immunogold and immunoperoxidase methods compared

The D2 neural cell adhesion molecule immunolabelling patterns of cultured bovine chromaffin cells are compared at the ultrastructural level, using the indirect immunohistochemical method with the secondary antibody labelled with either 17 or 5 nm gold particles or horseradish peroxidase (HRP). With HRP-conjugated antibody an apparently continuous surface staining was produced, while with 17 nm gold particles relatively large areas of membrane remained unlabelled; 5 nm particles gave a pattern more closely resembling the localization obtained with HRP. These differences are explained by differences in steric hindrance inherent to each method.     

Increased polysialic acid neural cell adhesion molecule expression in human hippocampus of heroin addicts

Chronic exposure to heroin is known to cause cognitive deficits. However, little is known about the underlying molecular mechanisms. It has been suggested that opiate-induced neurotoxicity as well as impaired plasticity and regeneration may be relevant. One of the target regions where regeneration still can be observed in the adult brain is the hippocampus. Since polysialic acid neural cell adhesion molecule is regarded as one of the key players involved in plasticity and regeneration of neural tissue, we analyzed polysialic acid neural cell adhesion molecule expression in the fascia dentate hilus of the human hippocampus of 29 lethally intoxicated heroin addicts and matched controls. Immunohistochemistry with an antibody directed against polysialic acid neural cell adhesion molecule revealed its expression in differently sized cells which could be identified as neurons and glial cells. We observed an increase in the percentage of polysialic acid neural cell adhesion molecule positive neurons in hippocampal hilus of heroin addicts compared with controls (P = 0.001).Interestingly, we also observed polysialic acid neural cell adhesion molecule expression in glial cells as evidenced by double immunofluorescence with glial fibrillary acidic protein and polysialic acid neural cell adhesion molecule using confocal laser scanning microscopy. The fraction of polysialic acid neural cell adhesion molecule positive glial cells was also higher in heroin addicts compared with controls (P = 0.009). In addition, within the group of addicts morphine blood concentrations showed a positive correlation with the percentage of polysialic acid neural cell adhesion molecule positive neurons (P = 0.04; r = 0.547). In conclusion, we observed an increase in polysialic acid neural cell adhesion molecule positive neurons and glial cells in hippocampi of heroin addicts. This might reflect an attempt to repair cell damage due to heroin exposure.     

神经细胞黏附分子1和骨调蛋白在神经管畸形中的变化

目的初步探究神经细胞黏附分子1和骨调蛋白在神经管畸形中的变化。方法20只健康成年SD孕大鼠随机分为正常对照组和神经管畸形实验组,每组10只。通过胃饲维甲酸建立胚胎大鼠神经管畸形模型,检查了神经管畸形鼠体重的变化;用HE染色观察神经管畸形鼠神经管结构的变化;通过Western blot分析神经管畸形鼠组织中神经细胞黏附分子1和骨调蛋白的变化。结果神经管畸形鼠形态偏小,体质量为(0.34±0.45)g,明显低于正常对照组(0.53±056)g。胚胎鼠在第9天神经管处于未闭合状态,在第11天已完全闭合。胚胎第9天和11天神经管畸形大鼠的神经细胞黏附分子1比正常组表达均明显增加(P0.01)。骨调蛋白在胚胎第9 d神经管畸形大鼠中的表达比正常明显增高(P0.01),但在胚胎第11天的表达与正常相比没有差异(P0.05)。结论神经细胞黏附分子1和骨调蛋白的变化可能与神经管畸形形成有密切关系。     

Structure and function of the gene for neural cell adhesion molecule

The neural cell adhesion molecule (NCAM) is a calcium-independent cell adhesion molecule that operates through a homophilic binding mechanism and is an important mediator of cell-cell interactions in a number of tissues including brain and skeletal muscle. The NCAM gene has been isolated and mechanisms for generating different NCAM proteins described. Alternative splicing allows the generation of more than twenty NCAM isoforms. In addition specific N- and O-linked carbohydrate modifications are likely to modulate NCAM function. Strategies are being developed to assess the role of NCAM isoforms and post-translational modifications by using gene transfer methods in conjunction with model assay systems. In this context, we will discuss the effects of polysialylation and alternative splicing on the ability of NCAM to function as a molecule that promotes neurite outgrowth.     

Gradient expression of neural cell adhesion molecule (NCAM) in the pontine migratory stream of fetal rats.

Immunohistochemical localization of a neural cell adhesion molecule (NCAM) was examined in the subpial pontine migratory stream of the fetal rat with a monoclonal antibody specific for the rat NCAM, MAb-AF11. Although the pontine cell strand showed weaker expression of NCAM than the parenchymal areas of the brainstem, within the cell strand, NCAM-immunoreactivity was somewhat greater in the anterior part of the stream near the neurons' destination than the caudal part. The weak NCAM gradient in the pontine migratory stream may contribute to formation of the pontine cell strand and the basal pontine gray.     

Developmentally regulated expression of the neural cell adhesion molecule (NCAM) by mouse thymocytes

The expression of the neural cell adhesion molecule (NCAM) has been investigated during thymus ontogeny. NCAM mRNA was readily detectable at day 19 of gestation, the youngest age studied. Its level declined after birth to become undetectable at 3 weeks of age. Cell surface expression of NCAM protein was detected on 14% of day 15 fetal thymocytes and peaked during the perinatal period, when around 40% of the thymocytes expressed low to medium levels of NCAM. At postnatal day 2, the vast majority of the NCAM+ cells were also CD4+ and CD8+. At embryonic day 15, NCAM appeared also to be expressed by CD4- thymocytes since 14% of the cells were already NCAM+ whereas CD4 was virtually undetectable. In frozen section of the newborn thymus, surface staining for NCAM was present on a subpopulation of cells in the cortex, rare in the medulla and absent from the sub-capsular area. In conjunction with other cell adhesion molecules, NCAM could play a role in cell interactions during thymic development.     

N-methyl-D-aspartate receptor blockade affects polysialylated neural cell adhesion molecule expression and synaptic density during striatal development.

Glutamatergic neurons innervate the striatum and form asymmetric synapses with the dendritic spines of striatal efferent neurons. The role of glutamate in striatal development, however, remains largely unknown. Previous studies have shown a dramatic increase in the density of asymmetric synapses in the rat striatum during the third postnatal week, followed by a decrease to adult levels by postnatal day 25. At the same time, the highly polysialylated form of the neural cell adhesion molecule becomes progressively restricted to synaptic regions and then disappears. We have now examined the effects of antagonists of the N-methyl-D-aspartate subtype of glutamatergic receptors on the expression of the polysialylated form of the neural cell adhesion molecule and on synaptic density during this late period of striatal development. Peripheral administration of the N-methyl-D-aspartate receptor antagonist dizocilpine maleate markedly decreased immunoreactivity for the highly polysialylated form of the neural cell adhesion molecule in the dorsolateral striatum and cerebral cortex when drug treatment included postnatal day 20, but not earlier in development. This effect was regionally specific and loss of the polysialylated neural cell adhesion molecule in the striatum was reproduced by the local administration of dizocilpine maleate, DL-2-amino-5-phosphonovalerate or ketamine on postnatal day 20. Quantitative ultrastructural studies of synaptic density with the physical disector method performed after one of the regimens inducing loss of the polysialylated neural cell adhesion molecule (postnatal days 18-20) revealed a 30% decrease in asymmetric synapses in the dorsolateral striatum of treated rats. Symmetric synapses, which presumably do not use glutamate, were not affected. The data indicate that N-methyl-D-aspartate receptors play a role in the late stages of synaptogenesis in the striatum and suggest that a subset of synapses expressing immunoreactivity for the highly polysialylated form of the neural cell adhesion molecule may be dependent on N-methyl-D-aspartate receptor stimulation during a critical period of striatal development.     

Expression and function of the neural cell adhesion molecule L1 in mouse leukocytes.

The neural cell adhesion molecule L1 is a cell surface glycoprotein of the immunoglobulin superfamily which mediates adhesion between neural cells. The possibility that similar cell-cell recognition mechanisms may be shared by the nervous and immune systems prompted us to study the expression and function of L1 in cells of the hematopoietic system. Immunofluorescence analysis using monoclonal L1 antibody revealed that the molecule is expressed in the bone marrow, spleen, and thymus of the mouse. This observation was confirmed by amplifying cDNA derived from these organs by the polymerase chain reaction with L1-specific oligonucleotide primers. Two-color fluorescence analysis indicated that bone marrow lymphoid and granulocyte precursor cells express low and high levels of L1, respectively. In the thymus L1 is primarily expressed by mature cells that have a strong expression of CD3 and in the spleen both B cells and T cells express L1. The possible function of L1 in lymphoid cells was studied using subcloned ESb-MP lymphoma cells having high or low densities of L1 on the cell surface as well as activated splenic B lymphoblasts. Parental and subcloned ESb-MP cells that strongly expressed L1 could form homotypic aggregates in the presence of low Ca2+ levels, whereas subcloned ESb-MP cells with a weak expression of L1 did not aggregate, suggesting that L1 mediates the Ca(2+)-independent aggregation of the parental ESb-MP cells. Furthermore, the aggregation of activated B lymphoblasts under physiological concentrations of Ca2+ and Mg2+ was inhibited by 30% in the presence of Fab fragments of polyclonal L1 antibodies, implying that L1 also mediates adhesion among normal lymphoid cells. A possible role of L1 on lymphocytes in stimulating the innervation of lymphoid organs is discussed.     

Expression of neural cell adhesion molecule in normal and neoplastic human neuroendocrine tissues.

The neural cell adhesion molecule (N-CAM) is a group of cell surface glycoproteins involved in direct cell--cell adhesion. N-CAM expression in normal and neoplastic tissues was examined with specific antibodies and oligonucleotide probes by immunohistochemistry and in situ hybridization. Most neuroendocrine cells and tumors with secretory granules expressed N-CAM protein and mRNA. Parathyroid adenomas (4) were somewhat unusual, because N-CAM mRNA, but not protein, was detected in some of these benign neoplasms. Most non-neuroendocrine cells and tumors did not express N-CAM, although uterine smooth muscle and an adrenal cortical carcinoma were both positive. Western blots disclosed proteins of 180, 140, and 120 kd in normal adult brain, whereas two pheochromocytomas, a null cell adenoma, and a gastrinoma had proteins of approximately 180 and 140 kd. These results indicate that N-CAM protein and mRNA are widely expressed in neuroendocrine cells and neoplasms. N-CAM oligonucleotide probes as well as antibodies against N-CAM can be used as broad-spectrum neuroendocrine markers. In addition, these molecular probes can be used to examine the role of N-CAM in the development and regulation of neuroendocrine tissues.     

Roles of immunoglobulin-like loops of junctional cell adhesion molecule 4; involvement in the subcellular localization and the cell adhesion

BACKGROUND: Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1) is a scaffolding protein at tight junctions (TJs). We have recently identified junctional adhesion molecule 4 (JAM4) as a MAGI-1-interacting protein. JAM4 belongs to the immunoglobulin superfamily and mediates Ca2+-independent adhesion. In this study, we examined the subcellular localization of JAM4 in various tissues and the involvement of JAM4 in the localization of MAGI-1. Moreover, we investigated into roles of immunoglobulin-like loops (Ig-loops) of JAM4. RESULTS: JAM4 was localized at TJs but also on apical membranes of epithelial cells in jejunum, ileum, and renal proximal tubules. In Madine Darby canine kidney (MDCK) cells, the localization of JAM4 at TJs depended on the first Ig-loop and did not require the MAGI-1-interacting region. JAM4 determined the subcellular localization of MAGI-1 in MDCK cells. In ileum, however, MAGI-1 was localized at TJs where JAM4 was not detected. Both of Ig-loops were necessary for homophilic interactions, but cis interactions depended on the first Ig-loop. CONCLUSION: JAM4 may be primarily targeted to apical membranes, and subsequently recruited to TJs through the first Ig-loop-mediated molecular interaction. JAM4 determines the localization of MAGI-1 in MDCK cells, but the in vivo localization of MAGI-1 does not necessarily depend on JAM4.     

Presence of the long chain form of polysialic acid of the neural cell adhesion molecule in Wilms'' tumor. Identification of a cell adhesion molecule as an oncodevelopmental antigen and implications for tumor histogenesis.

The long chain form of polysialic acid characteristic of the low adhesive embryonic form of the neural cell adhesion molecule NCAM is temporally and spatially expressed in developing kidney but undetectable in normal adult kidney. Therefore, this molecule represents a developmentally regulated antigen in kidney contrasted with neural tissue, where it is also detectable in the adult brain. This investigation of 25 Wilms' tumors comprising all different histologic types demonstrates expression of this molecule under conditions of malignant growth. Immunostaining was observed in Wilms' tumors with both a monoclonal anti-polysialic acid antibody and a polyclonal anti-NCAM polypeptide antiserum. Intense cell surface staining sensitive to endosialidases specifically hydrolyzing alpha 2,8 linked (poly)sialic acid was detectable in blastemal regions, and weaker, variable labeling was seen over tubules and glomeruloid bodies. The stroma was not stained. This is evidence indicating that Wilms' tumor originates from the embryonic equivalent of induced metanephrogenic mesenchyme. It seems unlikely however, that the stroma is derived from the blastema. The same high molecular mass broad band typical of the embryonic form of NCAM was revealed by immunoblot analysis of homogenates from Wilms' tumor as well as from embryonic kidney and brain. In situ hybridization demonstrated the presence of mRNA for NCAM in all but stromal elements of Wilm's tumors. Thus, polysialic acid is present on NCAM and represents a new oncodevelopmental antigen in human kidney. Polysialic acid was greatly reduced or absent by immunohistochemistry and immunoblotting in necrotic tumor areas.     

Effects of laboratory versus field exercise on leukocyte subsets and cell adhesion molecule expression in children

In adults, exercise is a powerful and natural stimulator of immune cells and adhesion molecules. Far less is known about these exercise responses during childhood and whether or not exercise in real-life activities of healthy children might influence immune responses. We compared laboratory exercise (10×2 min periods of heavy, constant intensity, cycle ergometer exercise with 1 min rests between exercise in nine subjects, aged 9–15 years) with field exercise (90 min soccer practice in nine different subjects, aged 9–11 years). Blood was sampled before both protocols, 5 min after the 30 min laboratory protocol, and 10–15 min after the 90 min field protocol. Both field and laboratory exercise protocols led to significant (P<0.05) increases in granulocytes, monocytes, and all lymphocyte subpopulations. The mean (SEM) increases were similar for the two protocols except for the significantly greater increase in laboratory compared with field protocols for natural killer cells [142 (39)% vs 12 (16)%, P<0.001] and monocytes [64 (22)% vs 32 (19)%, P<0.001]. Both protocols significantly influenced adhesion molecules (such as CD54) which have not been previously studied in children. However, the adhesion molecule CD8⁺CD62L^– increased to a significantly (P<0.001) greater extent in the laboratory [101 (25)%] versus field [34 (25)%] protocol. Finally, the density of CD62L on lymphocytes significantly decreased with laboratory exercise but showed no change in the field protocol [–20 (3)% vs –3 (3)%, P<0.001]. The rapid and substantial immune response in both laboratory and field protocols suggests that exercise stimulation of the immune system occurs commonly in the real lives of children and may play a role in their overall immune status. Electronic Publication